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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
Internationa} Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
G01N 27/42, 27/52, 27/49

(11) International Publication Number: WO 97/00441

(43) International Publication Date: 3 January 1997 (03.01 .97)

(21) International Application Number: PCT/AU96V00365

(22) International Filing Date: 19 June 1996 (19.06.96)

(30) Priority Data:

PN 3639

19 June 1995 (19.06.95)

(71) Applicant (for all designated States except US): MEMTEC

AMERICA CORPORATION [US/USJ; Suite 700, 9690
Dcereco Road, Timonium, MD 21093 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): HODGES, Alastair, Mchv
doe [AU/AU]; 34 Samuel Road, Blackburn South, VIC 3130
(AU). BECK, Thomas, William [AU/AU]; 31 Dmmmond
Street, South Windsor, NSW 2765 (AU). JOHANSEN, Odd-
var [NO/AU]; 16 Damley Grove, Mulgrave, VIC 3170
(AU).

(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY,
CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL,
IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV,
MD, MG, MK, MN, MW, MX. NO, NZ, PL, PT, RO, RU,
SD, SE, SG, SI, SK, 17, TM, TR, TT, UA, UG, US, UZ,
VN, ARIPO patent (KB, LS, MW, SD, SZ, UG), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published

With international search report.

(74) Agent: SHELSTON WATERS; 60 Margaret Street, Sydney,
NSW 2000 (AU).

(54) Title: ELECTROCHEMICAL CELL

(57) Abstract

A method for determining the concentration of a reduced (or oxidised) form of a redox species in an electrochemical cell of the
kind comprising a working electrode and a counter electrode spaced from the working electrode by a predetermined distance, said method
comprising the steps of: (1) applying an electric potential difference between the electrodes; (2) selecting the potential of the working
electrode such that the rate of electro-oxidation of the reduced form (or electro-reduction of the oxidised form) of the species is diffusion
controlled, (3) selecting the spacing between the working electrode and the counter electrode so that reaction products from the counter
electrode arrive at the working electrode; (4) determining current as a function of time after application of the potential and prior to
achievement of a steady state; (5) estimating the magnitude of the steady state current, and (6) obtaining from the change in current
with time and the magnitude of the steady state current, a value indicative of die diffusion coefficient and/or of the concentration of the
reduced form (or the oxidised form) of the species. Also disclosed is an apparatus for determining the concentration of a redox species
in an electrochemical cell comprising: an electrochemical cell having a working electrode and a counter (or counter/reference) electrode,
means for applying and electric potential difference between said electrodes, means for measuring the change in current with time, and
characterised in that the working electrode is spaced from the counter electrode by less than 500 jim.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCX

AM
AT

Armenia

United Kingdom

Austria

Georgia
Gufoea

All

Australia

Barbados

Greece

Belgium

Hungary

Burkina Paso

Ireland

Bulgaria

Italy

Benin

Japan

BR
BY

Brazil

Kenya

Belarus

Kyrgystan

Canada

Democratic People's Republic
of Korea

Central African Republic

Congo

Republic of Korea

Switzerland

Cote dlvoire

Cameroon

Sri Lanka

China

Liberia

Luxembourg

Latvia

Monaco

Estonia

Republic of Moldova

Spain

Madagascar

Finland

Mali

France

Mongolia

Gabon

Mauritania

Malawi

Mexico

Niger

Netherlands

Norway

New Zealand

Poland

Portugal

Romania

Russian Federation

SD
SE

Sudan
Sweden

Singapore

Slovenia

Slovakia

Senegal

Swaziland

Chad

Togo

Tajikistan

Trinidad and Tobago

Ukraine

Uganda

United States of America

Uzbekistan

Viet Nam

WO 97/00441

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PCT/AU96/00365

TITLE: ELECTROCHEMICAL CELL
FIELD OF THE INVENTION

This invention relates to a biosensor and more particularly to an electrochemical
biosensor for determining the concentration of an analyte in a carrier. The invention is
5 particularly useful for determining the concentration of glucose in blood and is described
herein with reference to that use but it should be understood that the invention is
applicable to other analytic determinations.
BACKGROUND OF THE INVENTION

Electrochemical biosensors generally comprise a cell having a working electrode,
10 a counter electrode and a reference electrode. Sometimes the function of the counter and
reference electrodes are combined in a single electrode called a "counter/reference"
electrode or "pseudo reference electrode". As herein used the term "counter electrode"
includes a counter/reference electrode where the context so admits.

The sample containing the analyte is brought into contact with a reagent
15 containing an enzyme and a redox mediator in the cell. Either the mediator is reduced

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(receives at least one electron) while the analyte is oxidised (donates at least one
electron) or visa versa. Usually it is the analyte which is oxidised and the mediator
which is reduced. The invention will be herein described principally with reference to
that system but it is also applicable to systems in which the analyte is reduced and the
5 mediator oxidised.

Electrochemical glucose analysers such as those used by diabetics to monitor
blood glucose levels or such as are used in clinics and hospitals are commonly based
upon the use of an enzyme such as glucose oxidase dehydrogenase (GOD) and a redox
mediator such as a ferricyanide or ferrocyanide. In such prior art system, the sample
10 (e.g. blood) containing the analyte (e.g. glucose) is brought into contact with the reagents
in the cell. Glucose is oxidised to gluconic acid and the glucose oxidase is thereby
reduced. The mediator then re-oxidizes the glucose oxidase and is reduced in the
process. The reduced mediator is then re-oxidized when it transfers electrons to the
working electrode. After allowing passage of a predetermined time, sufficient to obtain
15 an accurate estimate of the Faraday current, the concentration of glucose is estimated
from the magnitude of the current or voltage signal then measured.

Prior art electrochemical cells consist of two (or three) adjacent electrodes spaced
apart on one side of an insulator and adapted for connection to a measuring device. A
target area on which the blood sample is placed is defined on or between the electrodes.
20 Co-pending Application PCT/AU95/00207 describes a cell in which electrodes are
disposed on opposite sides of a porous membrane, one of the electrodes having a liquid
permeable target area.

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In the prior art there is a need to separate the working electrode from the counter
(or counter/reference) electrode by a sufficient distance to avoid products of
electrochemical reaction at one electrode from interfering with those at the other. In
practice a separation of the electrodes of more than 500 \im is required to achieve
5 acceptable acci^acy.

Each batch of cells is required to have been previously calibrated and leads to
inaccuracies during use because of variations within the batch, in sample composition,
and in ambient conditions.

It is desired to improve the accuracy and reliability of such biosensors.

1 0 Achievement of these objectives is made difficult in the case of sensors intended to

determine the concentration of analytes in blood because blood contains dissolved gases,
ions, colloids, complex micelles, small scale cellular debris, and living cellular
components in a predominantly aqueous medium; Any of these may interfere in the
determination. Existing sensors are also susceptible to influence from other interfering

1 5 substances that may be present in the sample and which may be oxidised at the working
electrode and mistakenly identified as the analyte of interest. Alternatively/the
interfering substances may reduce the oxidised form of the redox mediator. These
effects will give artificially elevated estimates of the analyte concentration. Additionally
there is always some reduced redox mediator present before the analyte is added and its

20 concentration needs to be known and subtracted from the measured value of reduced
mediator to give an accurate concentration of the analyte. Moreover, oxygen in the
blood may act as a redox mediator for glucose oxidase dehydrogenase (GOD) in
competition with ferrocyanide. Thus high oxygen concentrations can lead to low

SUBSTITUTE SHEET (RULE 26)

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estimates of glucose concentration. In addition the measurements are sensitive to factors
such as changes in humidity, temperature, solution viscosity and haematocrit content.
OBJECT OF THE INVENTION

It is an object of the present invention to provide a method of analysis and
5 apparatus for use in the method which avoid or ameliorate at least some of the
disadvantages of the prior art It is an object of preferred forms of the invention to
provide a biosensor of improved accuracy, and/or reliability and/or speed and a method
for its use.

DISCLOSURE OF THE INVENTION

According to one aspect the invention consists in a method for detennining the
concentration of a reduced (or oxidised) form of a redox species in an electrochemical
cell of the kind comprising a working electrode and a counter electrode spaced from the
working electrode by a predetermined distance, said method comprising the steps of:

(1) applying an electric potential difference between the electrodes,

(2) selecting the potential of the working electrode such that the rate of
electro-oxidation of the reduced form (or electro-reduction of the oxidised form) of the
species is diffusion controlled,

(3) selecting the spacing between the working electrode and the counter
electrode so that reaction products from the counter electrode arrive at the working

20 electrode,

(4) deterniining current as a function of time after application of the potential
and prior to achievement of a steady state,

(5) estimating the magnitude of foe steady state cuirent,and

SUBSTITUTE SHEET (RULE 26)

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(6) obtaining from the change in cuiTent with time and the magnitude of the
steady state current, a value indicative of the diffusion coefficient and/or of the
concentration of the reduced form (or the oxidised form) of the species.

The concentration measured in this way is substantially independent of variation
5 if any in the diffusion coefficient of the reduced form, and therefore is compensated for
variations in temperature and viscosity. The concentration so measured is independent
of variations in haematocrit and other substances which affect the diffusion coefficient of
the reduced form of the redox species.

It will be appreciated that the method of the invention is equally applicable for
1 0 determining the concentration of a reduced form of a redox species or an oxidized form
of a redox species in the cell. In the case that the concentration of the reduced form is to
be determined the potential of the working electrode must be maintained such that the
rate of electro oxidation of the reduced form is diffusion controlled in step (2) and it is
the concentration of the reduced form that is obtained in step (5). In the case that the
1 5 concentration of oxidized form is to be determined, the potential of the working

electrode must be maintained such that the rate of electro reduction of the oxidized form
is diffusion controlled in step (2) and it is the concentration of the oxidized form that is
obtained in step (5).

The redox species may be an analyte or may be a redox mediator.
20 In preferred embodiments of the method a mediator is used and the concentration

of the reduced (or oxidized) form of the mediator is in turn indicative of the
concentration of an analyte and a measure of the diffusion coefficient of the reduced (or

SUBSTITUTE SHEET (RULE 26)

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oxidized) form of the mediator is determined as a precursor to the determination of the
concentration of the analyte.

For preference the cell comprises a working electrode and counter/reference
electrode. If a reference electrode separate from a counter electrode is used, then the
5 reference electrode may be in any convenient location in which it is in contact with the
sample in the sensor.

In contrast to prior art, when conducting the method of the invention, the
electrodes are sufficiently close that the products of electrochemical reaction at the
counter electrode migrate to the working electrode during the period of the test For
10 example, in an enzyme ferricyanide system, the ferrocyanide produced at the counter
electrode diffuses to the working electrode.

This allows a steady state concentration profile to be achieved between the
electrodes leading to a steady state current This in turn allows the diffusion coefficient
and concentration of the redox species (mediator) to be measured independently of
15 sample variations and therefore greatly improves accuracy and reliability.

The method also permits the haematocrit concentration of blood to be determined
from the diffusion coefficient by use of look-up tables (or by separation of red cells from
plasma and measurement of the diffusion coefficient of the red cell fraction) and the
plasma fraction, and comparing the two.
20 According to a second aspect the invention consists in apparatus for determining

the concentration of a redox species in an electrochemical cell comprising:

an electrochemical cell having a working electrode and a counter (or
counter/reference) electrode,

SUBSTITUTE SHEET (RULE 26)

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means for applying an electric potential difference between said electrodes,
means for measuring the change in current with time,

and characterised in that the working electrode is spaced from the counter
electrode by less than 500 fim.
5 In preferred embodiments the cell has an effective volume of 1 .5 microlitres or

less. Apparatus for use in the invention may comprise a porous membrane, a working
electrode on one side of the membrane, a counter/reference electrode on the other side,
said electrodes together with a zone of the membrane therebetween defining an
electrochemical cell, and wherein the membrane extends laterally from the cell to a
10 sample deposition area spaced apart from the cell zone by a distance greater than the
thickness of the membrane.

Preferably the porous membrane, the distance of the target area from the cell
portion, and the membrane thickness are so selected in combination that when blood
(comprising plasma and red cells) is placed on the target area a plasma front diffuses
15 laterally towards the electrochemical celt zone in advance of the red cells.

It is thus possible to fill a thin layer electrochemical cell with plasma
substantially free of haematocrit which would cause a variation in the diffusion
coefficient of the redox mediator and which would affect the accuracy of the test as
hereinafter explained

20 In preferred embodiments of the biosensor according to the invention a second

electrochemical cell zone of the membrane is defined by a second working electrode and
a second counter/reference electrode on the opposite side of the membrane from the
second working electrode. The second electrochemical cell zone is situated intermediate

SUBSTITUTE SHEET (RULE 26)

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the first cell zone and the sample deposition or "target" area, or is situated on the side of
the target area remote from the first electrochemical zone. In these embodiments the
plasma comes into contact with enzyme in, or on route to, the first electrochemical cell
while plasma reaching the second cell does not. The first cell thus in use measures the
5 concentration of reduced mediator in the presence of plasma (including

electrochemically interfering substances), and enzyme while the second electrochemical
cell measures it in the presence of plasma (including electrochemically interfering
substances) and in the absence of enzyme. This allows determination of the
concentration of the reduced interfering substances in the second cell and the
9 concentration of reduced interfering substances plus analyte in the first cell. Subtraction
of the one value from the other gives the absolute concentration of analyte.

In a highly preferred embodiment of the invention a hollow cell is employed
wherein the working and reference (or counter/reference) electrodes are spaced apart by
less than 500 pm and preferably by from 20 - 200 um.
5 DESCRIPTION OF THE DRAWINGS

The invention will now be more particularly described by way of example only
with reference to the accompanying drawings wherein:

Figure 1 is a schematic drawing (not to scale) of a first embodiment according to
the invention shown in side elevation.
5 Figure 2 shows the embodiment of Figure 1 in plan, viewed from above.

Figure 3 shows the embodiment of Figure 1 in plan, viewed from below.
Figure 4 shows the embodiment of Figure 1 viewed in end elevation.

SUBSTITUTE SHEET (Rule 26)

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Figure 5 is a schematic drawing (not to scale) of a second embodiment according
to the invention in side elevation.

Figure 6 shows the embodiment of Figure 5 in plan, viewed from above.

Figure 7 is a schematic drawing (not to scale) of a third embodiment according to
5 the invention, in side elevation.

Figure 8 shows the embodiment of Figure 7 in plan, viewed from above.

Figure 9 is a schematic drawing (not to scale) according to the invention in plan
view, viewed from above.

Figure 10 shows the embodiment of Figure 9 in end elevation.
10 Figure 1 1 shows the embodiment of Figure 9 in side elevation.

Figure 12 shows a schematic drawing (not to scale) of a hollow cell embodiment
according to the invention, viewed in cross section.

Figure 13 is a graph showing a plot of current (ordinate axis) versus time (co-
ordinate axis) during conduct of a method according to the invention.
15 Figure 14 is a further graph of use in explaining the method of the invention.

In Figures 5 to 12, components corresponding in function to components of the
embodiment of Figures 1 to 4 are identified by identical numerals or indicia.
DESCRIPTION OF PREFERRED EMBODIMENTS

With reference to Figures 1 to 4 there is shown a first embodiment of apparatus
20 of the invention, in this case a biosensor for determining glucose in blood. The

embodiment comprises a thin strip membrane 1 having upper and lower surfaces 2, 3
and having a cell zone 4 defined between a working electrode 5 disposed on upper
surface 2 and a counter electrode 6 disposed on lower surface 3. The membrane

SUBSTITUTE SHEET (RULE 26)

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thickness is selected so that the electrodes are separated by a distance "1" which is
sufficiently close that the products of electrochemical reaction at the counter electrode
migrate to the working electrode during the time of the test and a steady state diffusion
profile is substantially achieved. Typically, T will be less than 500 urn. A sample
5 deposition or "target" area 7 defined on upper surface 2 of membrane 1 is spaced at a
distance greater than the membrane thickness from cell zone 4. Membrane 1 has a
diffusion zone 8 extending between target area 7 and cell zone 4. A suitable reagent
including a redox mediator "M", an enzyme "E" and a pH buffer "B" are contained
within cell zone 4 of the membrane and/or between cell zone 4 and target area 7. The
10 reagent may also include stabilisers and the like.

In some cases it is preferable to locate the enzyme and mediator and/or the buffer
in different zones of the membrane. For example the mediator may be initially located
within electrochemical cell zone 4 while the enzyme may be situated below target area 7
or in diffusion zone 8.

15 Haemoglobin releases oxygen at low pH's, but at higher pH's it binds oxygen

very firmly. Oxygen acts as a redox mediator for glucose oxidase dehydrogenase
(GOD). In a glucose sensor this competes with the redox mediator leading to low
estimates of glucose concentration. Therefore if desired a first pH buffer can be
contained in the vicinity of target area 7 to raise the pH to such a level that all the

20 oxygen is bound to haemoglobin. Such a pH would be non-optimal for GOD/glucose
kinetics and would consequently be detrimental to the speed and sensitivity of the test
In a preferred embodiment of the invention a second pH buffer is contained as a reagent
in the vicinity of the working electrode to restore the pH to kinetically optimal levels.

SUBSTITUTE SHEET (RULE 26)

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The use of a second buffer does not cause oxygen to be released from the haemoglobin
as the haemoglobin is contained within the blood cells which are retained near blood
target area 7 or are retarded in diffusion in comparison with the plasma and therefore not
influenced by the second buffer. In this manner oxygen interference may be greatly
5 reduced or eliminated.

In use of the sensor a drop of blood containing a concentration of glucose to be
determined is placed on target zone 7. The blood components wick towards cell zone 4,
the plasma component diffusing more rapidly than red blood cells so that a plasma front
reaches cell zone 4 in advance of blood cells.

10 When the plasma wicks into contact with the reagent, the reagent is dissolved and

a reaction occurs that oxidises the analyte and reduces the mediator. After allowing a
predetermined time to complete this reaction an electric potential difference is applied
between the working electrode and the counter electrode. The potential of the working
electrode is kept sufficiently anodic such that the rate of electro oxidation of the reduced

15 form of the mediator at the working electrode is determined by the rate of diffusion of
the reduced form of the mediator to the working electrode, and not by the rate of electron
transfer across the electrode/solution interface.

In addition the concentration of the oxidised form of the mediator at the counter
electrode is maintai n ed at a level sufficient to ensure that when a current flows in the

20 electrochemical cell the potential of the counter electrode, and thus also the potential of
the working electrode, is not shifted so far in the cathodic direction that the potential of
the working electrode is no longer in the diffusion controlled region. That is to say, the
concentration of the oxidized form at the counter electrode must be sufficient to maintain

SUBSTITUTE SHEET Otale 26)

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diffusion controlled electro oxidation of the reduced form of the mediator at the working
electrode.

The behaviour of a thin layer cell is such that if both oxidised and reduced forms
of the redox couple are present, eventually a steady state concentration profile is
5 established across the cell. This results in a steady state current It has been found that
by comparing a measure of the steady state current with the rate at which the current
varies in the current transient before the steady state is achieved, the diffusion coefficient
of the redox mediator can be measured as well as its concentration.

More specifically, by solving the difiusion equations for this situation it can be
10 shown that over a restricted time range a plot of ln(i/i" -1) vs time (measured in seconds)
is linear and has a slope (denoted by S) which is equal to -47t 2 D/l 2 , where "T is the
current at time "t", T " is the steady state current, "D M is the difiusion coefficient in
cm /sec, T is the distance between the electrodes in cm and V is approximately
3.14159. The concentration of reduced mediator present when the potential was applied
15 between the electrodes is given by 27t 2 r/FAl S, where "F" is Faraday's constant, A is the
working electrode area and the other symbols are as given above. As this later formula
uses S it includes the measured value of the diffusion coefficient

Since 1 is a constant for a given cell, measurement of i as a function of time and
f° enable the value of the diffusion coefficient of the redox mediator to be calculated and
20 the concentration of the analyte to be determined.

Moreover the determination of analyte concentration compensates for any
variation to the diffusion coefficient of the species which is electro oxidised or electro
reduced at the working electrode. Changes in the value of the diffusion coefficient may

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occur as a result of changes in the temperature and viscosity of the solution or variation
of the membrane permeability. Other adjustments to the measured value of the
concentration may be necessary to account for other factors such as changes to the cell
geometry, changes to the enzyme chemistry or other factors which may effect the
5 measured concentration. If the measurement is made on plasma substantially free of
haematocrit (which if present causes variation in the diffusion coefficient of the redox
mediator) the accuracy of the method is further improved.

Each of electrodes 5, 6 has a predefined area. In the embodiments of figures 1 to
4 cell zone 4 is defined by edges 9, 10, 1 1 of the membrane which correspond with edges

10 of electrodes 5, 6 and by leading (with respect to target area 7) edges 12, 13 of the
electrodes. In the present example the electrodes are about 600 angstrom thick and are
from 1 to 5 mm wide.

Optionally, both sides of the membrane are covered with the exception of the
target area 7 by laminating layers 14 (omitted from plan views) which serves to prevent

15 evaporation of water from the sample and to provide mechanical robustness to the

apparatus. Evaporation of water is undesirable as it concentrates the sample, allows the
electrodes to dry out, and allows the solution to cool, affecting the diffusion coefficient
and slowing the enzyme kinetics, although diffusion coefficient can be estimated as
above.

20 A second embodiment according to the invention, shown in Figures 5 and 6,

differs from the first embodiment by inclusion of a second working electrode 25 and
counter/reference electrode 26 defining a second cell zone 24 therebetween. These
electrodes are also spaced apart by less than 500 ^iminthe present example. Second

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electrodes 25, 26 are situated intermediate cell zone 4 and target area 7. In this
embodiment the redox mediator is contained in the membrane below or adjacent to
target area 7 or intermediate target area 7 and first cell zone 4. The enzyme is contained
in the membrane in the first cell zone 4 and second cell zone 24. The enzyme does not
5 extend into second cell 24. In this case when blood is added to the target area, it
dissolves the redox mediator. This wicks along the membrane so that second
electrochemical cell 24 contains redox mediator analyte and serum including
electrochemically interfering substances. First electrochemical cell receives mediator,
analyte, serum containing electrochemically interfering substances, and enzyme.

1 0 Potential is now applied between both working electrodes and the counter electrode or
electrodes but the change in current with time is measured separately for each pair. This
allows the determination of the concentration of reduced mediator in the absence of
analyte plus the concentration of electrochemically interfering substances in the second
electrochemical cell and the concentration of these plus analyte in the first

15 electrochemical cell. Subtraction of the one value from the other gives the absolute
concentration of analyte.

The same benefit is achieved by a different geometry in the embodiment of
Figures 7 and 8 in which the second working electrode and second counter/reference
electrode define the second cell 24 on the side of target area 7 remote from first

:o electrochemical cell 4. In this case the enzyme may be contained in the membrane strip
between the target area and cell 1. The redox mediator may be in the vicinity of the
target area or between the target area and each cell. The diffusion coefficient of
mediator is lowered by undissolved enzyme and the arrangement of Figures 7 and 8 has

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the advantage of keeping enzyme out of the thin layer cells and allowing a faster test (as
the steady state current is reached more quickly). Furthermore the diffusion constant of
redox mediator is then the same in both thin layer cells allowing more accurate
subtraction of interference.
5 Although the embodiments of Figures 1 to 8 are unitary sensors, it will be

understood that a plurality of sensors may be formed on a single membrane as shown in
the embodiment of Figures 9 to 1 1 . In this case the electrodes of one sensor are
conductively connected to those of an adjacent sensor. Sensors may be used
successively and severed from the strip after use.

10 In the embodiment of Figures 9 to 1 1 electrode dimensions are defined in the

diffusion direction (indicated by arrow) by the width of the electrode in that direction.
The effective dimension of the electrode in a direction transverse to diffusion direction is
defined between compressed volumes 16 of the membrane in a manner more fully
described in co-pending Application PCT/AU96/00210, the disclosures of which is

1 5 incorporated herein by reference in its entirety. For clarity optional laminated layer 1 4
of Figure 1 has been omitted from figures 9 to 1 1.

In the embodiment of Figure 1 2 there is shown a hollow cell according to the
invention wherein the electrodes 5, 6 are supported by spaced apart polymer walls 30 to
define a hollow cell. An opening 31 is provided on one side of the cell whereby a

20 sample can be admitted into cavity 32. In this embodiment a membrane is not used. As
in previous embodiments, the electrodes are spaced apart by less than 500 jim,
preferably 20 - 400 ym and more preferably 20 - 200 fim. Desirably the effective cell
volume is 1 .5 microlitres or less.

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It will be understood that the method of the invention may be performed with a
cell constructed in accord with co-pending application PCT/AU95/00207 or cells of
other known design, provided these are modified to provide a sufficiently small distance
between electrode faces.

5 The method of the invention will now be further exemplified with reference to

figures 13 and 14.
EXAMPLE 1

A membrane 130 microns thick was coated on both sides with a layer of
Platinum 60 nanometers thick. An area of 12.6 sq. mm was defined by compressing the
1 0 membrane. 1 .5 microlitres of a solution containing 0.2 Molar potassium ferricyanide
and 1 % by weight glucose oxidase dehydrogenase was added to the defined area of the
membrane and the water allowed to evaporate.

The platinum layers were then connected to a potentiostat to be used as the
working and counter/reference electrodes. 3.0 microlitres of an aqueous solution
15 contairring5niillimolre

area of the membrane. After an elapse of 20 seconds a voltage of 300 millivolts was
applied between the working and counter/reference electrodes and the current recorded
for a further 30 seconds at intervals of 0. 1 seconds.

Figure 13 is a graph of current versus time based on die above measurements.
20 Using a value of the steady state current of 26.9 microamps the function ln(i/26.9 - 1)
was computed and plotted versus time. The slope of die graph (Figure 14) is -0.342
which corresponds to a diffusion coefficient of 1.5 x 10" 6 cm 2 per second and a corrected
glucose concentration (subtracting background ferrocyanide) of 5.0 millimolar.

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The steady state current is one in which no further significant current change
occurs during the test. As will be understood by those skilled in the art, a minimum
current may be reached after which there may be a drift due to factors such as lateral
diffusion, evaporation, interfering electrochemical reactions or the like. However, in
5 practice it is not difficult to estimate the "steady state" current (f). One method for
doing so involves approximating an initial value for i 00 . Using the fit of the i versus t
data to the theoretical curve a better estimate of i 00 is then obtained. This is repeated
reiteratively until the measured value and approximated value converge to within an
acceptable difference, thus yielding an estimated i*

1 0 In practice, the measurements of current i at time t are made between a minimum

time t min and a maximum time t max after the potential is applied. The minimum and
maximum time are determined by the applicability of the equations and can readily be
determined by experiment of a routine nature. If desired the test may be repeated by
switching off the voltage and allowing the concentration profiles of the redox species to

1 5 return towards their initial states.

It is to be understood that the analysis of the current v. time curve to obtain
values of the Diffusion Co-efficient and/or concentration is not limited to the method
given above but could also be achieved by other methods.

For instance, the early part of the current v. time curve could be analysed by the

20 Cottrell equation to obtain a value of D* x Co (Co = Concentration of analyte) and the
steady state current analysed to obtain a value of D x Co. These 2 values can then be
compared to obtain D and C separately:

SUBSTITUTE SHEET fRule 2£\

WO 97/00441 PCT/AU96/00365

-18-

It will be understood that in practice of the invention an electrical signal is issued
by the apparatus which is indicative of change in current with time. The signal may be
an analogue or digital signal or may be a series of signals issued at predetermined time
intervals. These signals may be processed by means of a microprocessor or other
5 conventional circuit to perform the required calculations in accordance with stored
algorithms to yield an output signal indicative of the diffusion coefficient, analyte
concentration, haematocrit concentration or the like respectively. One or more such
output signals may be displayed by means of an analogue or digital display.

It is also possible by suitable cell design to operate the cell as a depletion cell
10 measuring the current required to deplete the mediator. For example in the embodiment
of Figure 5 the method of the invention may be performed using electrodes 5, 6, which
are spaced apart by less than 500 nm. An amperometric or voltammetric depletion
measurement may be made using electrodes 5 and 26 which are spaced apart more than
500 urn and such that there is no interference between the redox species being
1 5 amperometrically determined at electrodes 5, 26.

The depletion measurement may be made prior to, during or subsequent to, the
measurement of diffusion coefficient by the method of the invention. This enables a
substantial improvement in accuracy and reproducability to be obtained

In the embodiments described the membrane is preferably an asymmetric porous
20 membrane of the kind described in Patent No. 4,629,563 and 4,774,039 both of which
are incorporated herein in their entirety by reference. However symmetrical porous
membranes may be employed. The membrane may be in the form of a sheet, tube,
hollow fibre or other suitable form.

SUBSTITUTE SHEET (Rule 26)

WO 97/00441 PCT/AU96/00365

-19-

If the membrane is asymmetric the target area is preferably on the more open side
of the asymmetric membrane. The uncompressed membrane desirably has a thickness of
from 20 to 500 jim. The minimum thickness is selected having regard to speed,
sensitivity, accuracy and cost. If desired a gel may be employed to separate haematocrit
5 from GOD. The gel may be present between the electrodes and/or in the space between
the sample application area and the electrodes.

The working electrode is of any suitable metal for example gold, silver, platinum,
palladium, iridium, lead, a suitable alloy. The working electrode may be preformed or
formed in situ by any suitable method for example sputtering, evaporation under partial

10 vacuum, by electrodeless plating, electroplating, or the like. Suitable non-metal
conductors may also be used for electrode construction. For example, conducting
polymers such as poly(pyrrole), poly(aniline), porphyrin "wires", poly(isoprene) and
poly (cis-butadiene) doped with iodine and "ladder polymers". Other non-metal
electrodes may be graphite or carbon mixed with a binder, or k carbon filled plastic.

15 Inorganic electrodes such as In 2 0 3 or Sn0 2 may also be used. The counter/reference
electrode may for example be of similar construction to the working electrode. Nickel
hydroxide or a silver halide may also be used to form the counter/reference electrode.
Silver chloride may be employed but it will be understood that chloridisation may not be
necessary and silver may be used if sufficient chloride ions are present in the blood

20 sample. Although in the embodiments described the working electrode is shown on the
upper surface of the biosensor and the counter/reference electrode is on the lower
surface, these may be reversed.

STIRSTITUTH SHEET fltale 26)

WO 97/00441 PCT/AU96/00365

-20-

It is preferable that the working electrode and counter (or counter/reference)
electrodes are of substantially the same effective geometric area.

If a separate reference and counter electrode are employed, they may be of
similar construction. The reference electrode can be in any suitable location.
5 It will be understood that the features of one embodiment hereindescribed may be

combined with those of another. The invention is not limited to use with any particular
combination of enzyme and mediator and combinations such as are described in EP
0351 892 or elsewhere may be employed. The system may be used to determine analytes
other than glucose (for example, cholesterol) by suitable adaptation of reagents and by
1 0 appropriate membrane selection. Thje system may also be adapted for use with media
other than blood. For example, the method may be employed to determine the
concentration of contaminants such as chlorine, iron, lead, cadmium, copper, etc., in
water.

Although the cells herein described have generally planar and parallel electrodes
15 it will be understood that other configurations may be employed, for example one
electrode could be a rod or needle and the other a concentric sleeve.

It will be apparent to those skilled in the art from the disclosure hereof the
invention may be embodied in other forms without departing from the inventive concept
herein disclosed.

SUBSTITUTE SHEET (RULE 26)

WO 97/00441 PCT/AU96/00365

-21-

CLAIMS:

1 . A method for determining the concentration of a reduced (or oxidised) form of a
redox species in an electrochemical cell of the kind comprising a working electrode and
a counter electrode spaced from the working electrode by a predetermined distance, said

5 method comprising the steps of:

(1) applying an electric potential difference between the electrodes,

(2) selecting the potential of the working electrode such that the rate of
electro-oxidation of the reduced form (or electro-reduction of the oxidised form) of the
species is diffusion controlled,

1 0 (3) selecting the spacing between the working electrode and the counter

electrode so that reaction products from the counter electrode arrive at the working
electrode,

(4) determining current as a function of time after application of the potential
and prior to achievement of a steady state,
15 (5) estimating the magnitude of the steady state current, and

(6) obtaining from the change in current with time and the magnitude of the
steady state current, a value indicative of the diffusion coefficient and/or of the
concentration of the reduced form (or the oxidised form) of the species.

2. A method according to Claim 1 wherein a steady state concentration profile of
20 electrochemical reaction products is achieved between the working electrode and counter

electrode during the period of a test.

3. A method according to Claim 1 or Claim 2 wherein the electrodes are separated
by less than 500 \xm.

SUBSTITUTE SHEET (RULE 26)

WO 97/00441 PCT/AU96/00365

-22-

4. A method according to any one of the preceding claims wherein the redox
species is a mediator and the concentration of the reduced (or oxidised) form of the
mediator is indicative of the concentration of an analyte and wherein a measure of the
diffiision coefficient of the reduced (or oxidised) form of the mediator is determined as a

5 precursor to the determination of the concentration of the analyte.

5. A method according to any one of the preceding claims wherein the steady state
current is estimated by approximating an initial value for the steady state current,
measuring a discrepancy between measured current versus time data and a theoretical
curve, and using the degree of discrepancy, if any, to obtain a better estimate of the

10 steady state current

6. A method according to any one of claims 1 to 3 wherein the redox species is an
analyte.

7. A method according to any one of claims 1 to 3 wherein the redox species is a
mediator.

15 8. A method according to any one of the preceding claims wherein the cell

comprises a working electrode, a counter electrode and a separate reference electrode.
9. A method according to any one of the preceding claims wherein the spacing
between the electrodes is selected such that the steady state current is achieved within a
desired time.

20 10. A method substantially as herein described with reference to any one of the
Examples.

11. Apparatus for use in a method according to Claim 1 comprising a porous
membrane, a working electrode on one side of the membrane, a counter electrode on the

SUBSTITUTE SHEET (RULE 26)

WO 97/00441 PCT/AU96/00365

-23-

other side of the membrane, said electrodes together with a zone of the membrane
therebetween defin^g an electrochemical cell, and wherein the membrane extends
laterally from the cell to a sample deposition area spaced apart from the cell zone by a
distance greater than the thickness of the membrane.
5 1 2. Apparatus according to Claim 1 1 wherein the electrochemical cell contains a
reagent

13. Apparatus according to Claim 1 1 or Claim 12 wherein the electrochemical cell
contains a mediator.

1 4. Apparatus according to any one of claims 1 1 to 1 3 wherein the membrane

1 0 contains an enzyme intermediate the electrochemical cell and the sample deposition area.

15. Apparatus according to any one of claims 1 1 to 1 4 further comprising a pH
buffer in the vicinity of the sample deposition area.

1 6. Apparatus according to Claim 1 5 further comprising a pH buffer in the vicinity
of the working electrode.

15 17. Apparatus according to Claim 1 1 comprising a second electrochemical cell zone
of the membrane defined by a second working electrode and a second counter/reference
electrode on the opposite side of the membrane from the second working electrode,
1 8. Apparatus according to Claim 17 wherein the second electrochemical cell zone is
situated intermediate the first cell zone and the sample deposition or target area.

20 1 9. Apparatus according to any one of claims 1 1 to 1 8 wherein the uncompressed
membrane has a thickness of less than 500 jim.

SUBSTITUTE SHEET (RULE 26)

WO 97/00441 PCT/AU96/00365

-24-

20. Apparatus according to any one of claims 1 1 to 19 wherein the working electrode
is a metal selected from the group comprising gold, silver, platinum, palladium, iridium,
lead and alloys thereof.

21 . Apparatus for determining the concentration of a redox species in an
5 electrochemical cell comprising:

an electrochemical cell having a working electrode and a counter (or
counter/reference) electrode,

means for applying an electric potential difference between said electrodes,
means for measuring the change in current with time, and characterised in that
10 the working electrode is spaced from the counter electrode by less than 500 um.

22. A hollow electrochemical cell comprising a working electrode, a counter and an
opening for admitting an analyte to the cell, the working electrode being spaced from the
counter by less than 500 um.

23. An electrochemical cell according to claim 21 or claim 22 wherein the electrodes
15 are spaced from 100 - 200 um apart.

24. An electrochemical cell according to any one of claims 21 to 23 wherein the
electrodes are facing one another.

25. An electrochemical cell according to any one of claims 21 to 24 wherein the
electrodes are of substantially corresponding area.

20 26. An electrochemical cell according to any one of claims 21 to 25 comprising a
working electrode, a counter electrode and a separate reference electrode.
27. An electrochemical cell according to any one of claims 21 to 26 having an
effective cell volume of less than 1 .5 microlitres.

SUBSTITUTE SHEET (RULE 26)

WO 97/00441 PCT/AU96/00365

-25-

28. Apparatus substantially as herein described with reference to any one of the
Examples.

29. Apparatus according to any one of claims 1 1 to 28 for use in measuring the
concentration of glucose in blood.

5 30. A method according to any one of claims 1 to 1 0 when used for determining
haematocrit concentration.

SUBSTITUTE SHEET (RULE 26)

WO 97/00441

PCT/AU96/00365

1/5

FIOA

SUBSTITUTE SHEET (RULE 26)

PCT/AU96/0036S

SUBSTITUTE SHEET (Rule 2ffi

WO 97/00441

PCT/AU96/00365

~r~n

F/G.10

15 25 5

]

26 6

FIG. 11

30
I

' \ ((((<< A

32 n

A V V V,

FIG 12

SUBSTITUTE SHEET (RULE 26)

WO 97/00441

PCT/AU96/00365

4/5

(sdweojDjui) judjjnj

SUBSTITUTE SHEET (RULE 26)

WO 97/00441

PCT/AU96/00365

5/5

•

--S3

-5

| 1 1 1 1 1 , 1 — j — +_

i w »: v (\j (O cO cO i
i i i i i i i i i

tl-i»H/l)U|

SUBSTITUTE SHEET (Rule 26)

INTERNATIONAL SEARCH REPORT

International Application No.
PCT/AU 96/00365

CLASSIFICATION OF SUBJECT MATTER

Int Cfi : G01N 27/42 27/52 27/49

According to International Patent Classification (IPC) or to both national classification and IPC

FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)
IPC :G01N 27/42 27/52 27/49

Documentation searched other than nunimura documentation to the extent that such documents are included in the fields searched
AU : IPC as above

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

X
A

EP 255291 A (UNILEVER NV) 3 February 1988
column 3 lines 20-58
the whole document

WO 94/02842 A (THE VICTORIA UNIVERSITY OF MANCHESTER)
3 February 1994
the whole document

AU 3 1042/93 A (COMMONWEALTH SCIENTIFIC AND INDUSTRIAL
RESEARCH ORGANISATION) 15 Jury 1993
the whole document

11-29
M0, 30

1-30

Further documents arc listed in the continuation of Box C

CHI

See patent family a

"O"

Special categories of cited documents:

document defining the general state of the art which is
not considered to be of particular relevance
earlier document but published on or after the

document which may throw doubts on priority claim(s)
or which is cited to establish the publication date of
another citation or other special reason (as specified)
document referring to an oral disclosure, use,
exhibition or other means

document published prior to the international filing
date but later than the priority date claimed

T later document published after the international riling date or
priority date and not in conflict with the application but cited to
understand the principle or theory underlying the invention

"X" document of particular relevance; the claimed invention cannot
be considered novel or cannot be considered to involve an
inventive step when the document is taken alone

*Y" document of particular relevance; the claimed invention cannot
be considered to involve an inventive step when the document is
c ombin ed with one or more other such documents, such
combination being obvious to a person skilled in the art

"&* document member of the same patent family

Date of the actual completion of the international search
9 August 1996

Date of mailing of the international search report

28 AU6W96

Name and mailing address of the 1SA/AU

AUSTRALIAN INDUSTRIAL PROPERTY ORGANISATION

PO BOX 200

WODEN ACT 2606

AUSTRALIA Facsimile No.: (06) 285 3929

Authorized officer

Z> STANOJEVIC

Telephone No.: (06) 283 2168

Form PCT/ISA/2 10 (second sheet) (July 1 992) cophin

Farm PCT/ISA/210 (continuation of second sheet) (July 1992) copirin

INTERNATIONAL SEARCH REPORT

international Application No.
PCT/AU 96/00365

Box 1 Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)

This International Search Report has not been established in respect of certain claims under Article 17(2)(a) for the following

reasons:

I. Claims Nos.:

because they relate to subject matter not required to be searched by this Authority, namely:

2. J J Claims Nos.:

because they relate to parts of the international application that do not comply with the prescribed requrrements to
such an extent that no meaningrul international search can be carried out, specifically:

3. |~J Claims Nos.:

because they are dependent claims and are hot drafted in accordance with the second and third sentences of Rule
6.4(a)

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

1 . Claims 1-10, 30 directed to a method for deterrruning the concentration of a reduced (or oxidised) form of a redox
species in an electrochemical cell.

2. Claims 1 1-20, 28, 29 directed to an apparatus for use in a method according to claim 1.

continued on the extra sheet

1 0 As 311 required additional search fees were timely paid by the applicant, this international search report covers all
searchable claims

2 ' [ill ^ 311 searchable claims could be searched without effort justifying an additional fee, this Authority did not invite
payment of any additional fee.

3. As only some of the required additional search fees were timely paid by the applicant, this international search

report covers only those claims for which fees were paid, specifically claims Nos. :

I I No required additional search fees were timely paid by the applicant ConsequenUy, this international search
report is restricted to the invention first mentioned in the claims; it is covered by claims Nos. :

Remark on Protest | | The additional search fees were accompanied by the applicant's protest

| | No protest accornpanied the payment of additional search fees.

Form PCT/KA/210 (continuation of first sheet(l)) (July 1992) cophin

INTERNATIONAL SEARCH REPORT

International Application No.
PCT/AU 96/00365

3. Claims 2 1-27 directed to an apparatus
as reasoned below:

concentration of a redox species in an electrochemical cell,

The international application does not comply with the requirements of unity of invention because h does not relate t«
one invention or to a group of invention so linked as to form a single general inventive concStacZL^ ST
conclusion the International Searching Authority has found that Zeara threTuSoS ^ "*

1-10, 30 directed to a memri^^
redox species comprising sbc different steps including the steps of detemuning current as a function of time and

2. Claims 11-20, 28 29 directed to an apparatus "foruse-mantethodac^rdingtoclaim 1. It is considered that
nTJT an STIk T to ^^^^thusthe claims are directed toTa^nT
having ^ekctrochenucal cell defined by a porous membrane, a working electrode and a counter eleSodTft is

** " electrochemical «« **** ^ a porous membrane, a working electrode aTa co\mte7
electrode comprises a second "special technical feature". ana a counter

3. Claims 2l " 27 ^t^to an apparatus for determining the concentration of a redox species in an dectrocfaemical

u " ^ ^ deCtr0de * si*** the counter electrode byTess than 500 uT

*^«Stec^cS^^re^ e ' ectro( k s P ace< ' fr 0 ™ ^ counter electrode by less than 500 pm comprisesa third

nSo!S,S > ^ enti ^ ed ° fC,aimS d0 1101 Share either ofthe technical features identified, a "technical
relationship between the inventus, as defined in PCT rule 13.2 does not exist. Accordingly t^uiten^ond anphcation
does not relate to one invention or to a single inventive concept. «™anonai application

Fonn PCT/ISA/210 (extra sheet) (July 1992) cophin

INTERNATIONAL SEARCH REPORT
Information on patent family members

International Application No.
PCT/AU 96/00365

This Annex lists the known " A* publication level patent family members relating to the patent documents cited
in the above-mentioned international search report. The Australian Patent Office is in no way liable for these
particulars which are merely given for the purpose of information.

Patent Document Cited in Search
Report

Patent Family Member

255291

470649

ES 2033854

9402842

47160/93

31042/93

Form PCT/ISA/210 (patent family annex) (July 1992) copfem

END OF ANNEX

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